The present invention relates to a drug delivery device comprising eukaryotic cells that contain an amplified expression vector. The drug delivery device of the invention is designed to provide physiological activities lacking in disease conditions or antagonists against conditions in an animal, especially a human.
The ability of eukaryotic cells to synthesize molecules results from a variety of metabolic activities which are provided by a variety of cellular factors, for example growth factors, hormones and enzymes. These metabolic activities are often part of metabolic pathways and aberrant control or a partial or complete lack of such an activity in an animal may be responsible for a disease condition in that animal, for example a human. Such metabolic activities may relate to energy metabolism, structural metabolism, signal transduction, or any kind of biochemical activity.
Attempts to restore or inhibit a metabolic activity to a diseased animal, including a human, have been made through gene therapy, i.e., through introduction of foreign polynucleotide sequences encoding agonists or antagonists into the genome of a cell in an animal. When gene therapy is carried out directly on an animal, it is difficult to control the outcome of the introduction of such sequences. For example, the foreign polynucleotide sequences may disrupt a cellular gene and thus lead to serious pathological conditions (e.g., cancer). Another problem often encountered in gene therapy is that the foreign polynucleotide sequences do not remain stably integrated and expressed in the target cells.
Gene therapy can be rendered much more controlled and effective if it is carried out in cultured cells which are then transferred into the diseased animal. However, in order to avoid the rejection of the manipulated cells that are transferred into an animal by its immune system, it is necessary to use autologous cells, derived from the treated animal itself. Alternatively, the manipulated cells may be from a different animal as the diseased animal (and possibly from a different species), thus requiring efforts to prevent rejection of the transferred cells through the immune system of the recipient animal.
One approach to prevent the rejection of foreign cells in an animal is to encapsulate such cells. By incorporating the cells into a protective shell, i.e., capsule, which prevents the immune system of the recipient animal to reach the cells, the cells can survive and remain metabolically active in the recipient animal. Critical to such survival is the fact that the capsule allows the passage of small molecules in and out of the capsule, thus providing the encapsulated cells with access to nutrients and the recipient animal to metabolic products of the encapsulated cells. As one or more of these metabolic products of the encapsulated cells is lacking in the recipient animal, the encapsulated cells effectively act as a drug pump.
A limitation when using such an encapsulation approach is that the capsules can take up only a limited number of cells and that not all cells within a capsule will survive over extended periods of time. In order to supply an effective level of the desired metabolic activity to the recipient animal, it is necessary to express such metabolic activity at a high level over extended periods of time in the encapsulated cells. A need exists for ways to make drug delivery through encapsulated cells more potent. The present invention provides such technology.
The present invention provides a drug delivery device (xe2x80x9cdevicexe2x80x9d) and methods of making and using such a device. The device of the present invention is useful for many disease control and therapeutic strategies by taking advantage of the ability of a cell to provide pharmaceutically useful functions, for example the metabolism of a drug precursor. Thus, the device of the invention can, once transferred into a diseased animal, provide long term disease control.
In one embodiment, the device of the present invention comprises a eukaryotic cell that contains an exogenous polynucleotide (xe2x80x9cexogenous polynucleotidexe2x80x9d) sequence, preferably in multiple copies, and that is located in a capsule. In one aspect, the exogenous polynucleotide comprises an amplification-promoting sequence and an expression cassette. In another aspect, the exogenous polynucleotide also comprises a selectable marker.
In one embodiment, an amplification-promoting sequence (xe2x80x9cAPSxe2x80x9d) useful for the exogenous polynucleotide in a device of the present invention, is capable of amplifying the number of copies of the exogenous polynucleotide in a eukaryotic cell. In a preferred aspect, an APS presented in SEQ ID NO:1 or SEQ ID NO:2 is used. In a further aspect, any polynucleotide that is substantially homologous to SEQ ID NOS:1 or 2 is also a useful APS in the exogenous polynucleotide. In another aspect, more than one APS is included in the exogenous polynucleotide, for example SEQ ID NOS:1 and 2. In a further aspect, one or more APSs are included in the exogenous polynucleotides, of which one or more is substantially homologous to either SEQ ID NOS:1 or 2 and of which one or more is identical to SEQ ID NOS:1 or 2.
An expression cassette useful in the exogenous polypeptide, in one embodiment, comprises a polynucleotide sequence that encodes a function of interest (xe2x80x9ccoding sequencexe2x80x9d) and a regulatory sequence. The coding sequence in the expression cassette, in one aspect, may encode a peptide, a polypeptide, a protein, a polynucleotide. In another aspect, the expression cassette may be specific for a polynucleotide or an oligonucleotide with a catalytic or an inhibitory activity. Preferably, the coding sequence is specific for a molecule that has a pharmacologically desirable activity or property. In another aspect, the coding sequence encodes a protein or a polypeptide with an enzyme activity that is lacking in a disease condition, for example an enzyme activity involved in the synthesis of a signal transduction molecule (e.g., a second messenger, serotonin, dopamin). In another aspect, the coding sequence is specific for a peptide, a polypeptide or a protein capable of regulating cell proliferation, cell differentiation, programmed cell death, signal transduction, gene expression, gene transcription, translation, etc. In another aspect the coding sequence is specific for aft antibody, an antibody fragment, an agonist of a naturally occurring molecule, an antagonist of a naturally occurring molecule, a neurotrophic factor, a neurotropic factor, a peptide hormone (e.g., a neurohormone), etc.
In another preferred embodiment, the coding sequence is specific for tyrosine hydroxylase, tryptophan hydroxylase, melanoma inhibitory activity, an insulin, an insulin precursor polypeptide, an enkephalin, an enkephalin precursor polypeptide, parathyroid hormone, a parathyroid hormone precursor polypeptide, enkephalinase, a neurotrophic factor, a neurotropic factor, or a homolog, orthologue, paralogue, analogue, or derivative of any of these, or a fusion polypeptide or protein that contains any of the above and a heterologous protein, polypeptide or peptide.
Preferably, the expression cassette contains a regulatory sequence. In one aspect, the expression cassette contains a promoter sequence and a polyadenylation sequence (xe2x80x9cpolyA sequencexe2x80x9d). Promoter sequences useful for the expression cassette, in a preferred aspect, are active, i.e., capable of initiating gene transcription, in the cell type used in the device of the present invention. Useful promoters are active at least at a moderate level (i.e., a moderately strong promoter), preferably they are active at a high level (i.e., strong promoter). A constitutive and an inducible promoter may be used in the expression cassette, provided the promoter is sufficiently active. In another aspect, any polyA sequence that is sufficiently strong to induce polyadenylation of the transcripts of the expression cassette can be used.
In another aspect, the expression cassette may include an enhancer, an intron, a 5xe2x80x2 and/or 3xe2x80x2 untranslated region, an RNA stability regulating sequence or a combination of more than one of these elements.
In one embodiment, a eukaryotic cell is used in the device of the present invention. The cell preferably is capable of dividing multiple times, for example about 10 to about 300 times, or more than 300 times. In one aspect, the cell is transformed. In another aspect, the cell is derived from the same species as the species in which the device of the present invention is used.
In one embodiment, the cell containing the exogenous polynucleotide is encapsulated. In a preferred aspect, the capsule that contains the cell is double layered. In a preferred aspect, the cells of the device of the present invention are located in the inner layer. In another aspect, the capsule has more than two layers.